It is widely known that circulating tumor cells arrest in the microvasculature, but this arrest is not random. For example, breast cancer cells preferentially arrest in the small blood vessels of the lungs, liver, brain and bones. The underlying mechanisms responsible for this preferential arrest of breast cancer cells in distant organs are not well understood. The long-term goal of our research is to elucidate the relationships between microcirculation-induced mechanical factors, microvascular permeability (vascular integrity), cell adhesion molecules, nitric oxide and cytokines, and tumor metastasis in intact microvessels. The objective of this project is to investigate the relationships between localized shear rates and stresses in curved/stretched microvessels, VEGF (vascular endothelial growth factor)-induced microvascular hyperpermeability, and mammary tumor cell arrest and adhesion in intact microvessels. On the basis of our preliminary studies, we shall use a newly developed in vivo single vessel perfusion/bending method that can create non-uniformly distributed shear rates/stresses along the vessel wall to test two hypotheses: 1) Tumor cells prefer to arrest at the locations of higher shear rates/stresses and shear rate/stress gradients in the post-capillary venules of microvasculature. The higher shear rates/stresses and shear rate/stress gradients activate the endothelial cells and the tumor cells (specifically, activate cell adhesion molecules and endothelial nitric oxide synthase) to increase the binding of tumor cells to the vessel wall and to increase the accumulation of tumor cells; 2) Tumor cells prefer to arrest in the microvessel with the increased permeability. The increased tumor cell adhesion to the microvessel wall with increased permeability is partially due to the radial pressure gradient that drives the cells towards the wall. These ideas will be explored using a combination of physiological, biochemical, mathematical and imaging approaches. Specific aims are: 1) use quantitative fluorescence video and confocal microscopy to determine the adhesion rates of normal, non-malignant (MCF-10A), and malignant (AU-565) breast epithelial cells in straight and curved/stretched microvessels on rat mesentery under known bulk flow rates and a) under conditions of normal and increased permeability by VEGF, b) after pretreatment with the blocking antibodies to endothelial cell adhesion molecules, c) after pretreatment with the blocking antibodies to tumor cell adhesion molecules and d) after pretreatment with eNOS inhibitors to microvessel endothelial cells; 2) use filter-based adhesion/transmigration assays to determine the adhesion/transmigration rates of above cells to/across cultured cell monolayers of microvascular endothelial cells isolated from the lung, brain, kidney and muscle under the same conditions as in Aim 1; 3) use fluorescence video and confocal microscopy to quantify the nitric oxide production in straight and curved/stretched microvessels under various bulk flow rates and under the same conditions a and d in Aim 1, and in cultured cell monolayer of lung and brain, kidney glomerulus and skeleton muscle microvascular endothelial cells under the same conditions a and d in Aim 1; and 4) quantify the shear rate, shear stress, normal stress (pressure), velocity and vorticity profiles by numerical simulation in the straight and curved/stretched microvessels under known bulk flow rates and under the conditions of normal and increased permeability by VEGF. PUBLIC HEALTH RELEVANCE: This project will lead to a quantitative understanding of the role of hydrodynamic factors, cell adhesion molecules and nitric oxide in tumor preferential metastasis, and hence help define a new class of targets for therapeutic drug design for cancer. We hope that inhibitory reagents that prevent cancer cell arrest and adhesion in the microcirculation and reagents that enhance the microvessel wall integrity may be used in combination with traditional therapies to combat this malignant disease more effectively.